Ca2+ current flowing via sarcolemmal Ca2+ channels of heart cells is an important determinant of cell Ca2+ homeostasis and thus an important determinant of a variety of cell functions. While Ca2+- and membrane voltage-dependent positive regulation of Ca2+ channels have been recently reported, little is known of the mechanism. Our studies provide compelling evidence that in adult rat cardiac ventricular cells Ca2+/calmodulin dependent kinase II mediates L-type Ca2+ current facilitation, i.e., an augmentation in current magnitude and or slowing of its inactivation during repetitive depolarizations, by single strong depolarizing prepulse or by depolarizing holding potentials. All of these effects on the current were completely abolished by a inclusion of specific peptide inhibitor of Ca2+/calmodulin kinase II within the pipette filling solution or by the replacement of Ca2+ with Ba2+ in bath solution. The involvement of Ca2+/calmodulin kinase II on Ca2+ current regulation is further supported by the localized distribution of a specific antibody to the kinase near cell sarcolemma as revealed by digital confocal fluorescent imaging. Furthermore, the immunofluorescence of the antibody was significantly enhanced by depolarization with high [K+]o and attenuated by removal of Ca2+o or by W7, a calmodulin inhibitor. Thus, Ca2+/calmodulin kinase II dependent protein phosphorylation is an important mechanism by which variable factors, such as Ca2+, repetitive stimulation and strong depolarization can positively regulate Ca2+ current in heart cells, and that membrane potential plays an essential role in the modulation of the kinase activity. These findings provide new insights toward understanding Ca2+ channel regulation in cardiac cells as possibly in other cell types as well.